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Tetrameric voltage-gated K + channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K + channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel at physiological voltages by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the KCNQ1-KCNE3-calmodulin complex structure, we thoroughly surveyed amino acid residues on KCNE3 and the S1 segment of the KCNQ1 voltage sensor facing each other. By changing the side-chain bulkiness of these interacting amino acid residues (volume scanning), we found that the distance between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of KCNQ1 and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests that tight binding of the S1 segment and KCNE3 is crucial for controlling the voltage sensor domains.

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart 1 , 2 . Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary β-subunits—intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)—to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials 1 – 5 . However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2–DPP6S–KChIP1 dodecamer complex, the Kv4.2–KChIP1 and Kv4.2–DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2–KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1–S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2–KChIP1–DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex. Cryo-electron microscopy structures of the voltage-gated potassium channel Kv4.2 alone and in complex with auxiliary subunits (DPP6S and/or KChIP1) reveal the distinct mechanisms of these two different subunits in modulating channel activity.

Voltage‐gated K+ (KV) and Ca2+‐activated K+ (KCa) channels are essential proteins for membrane repolarization in excitable cells. They also play important physiological roles in non‐excitable cells. Their diverse physiological functions are in part the result of their auxiliary subunits. Auxiliary subunits can alter the expression level, voltage dependence, activation/deactivation kinetics, and inactivation properties of the bound channel. KV and KCa channels are activated by membrane depolarization through the voltage‐sensing domain (VSD), so modulation of KV and KCa channels through the VSD is reasonable. Recent cryo‐EM structures of the KV or KCa channel complex with auxiliary subunits are shedding light on how these subunits bind to and modulate the VSD. In this review, we will discuss four examples of auxiliary subunits that bind directly to the VSD of KV or KCa channels: KCNQ1–KCNE3, Kv4‐DPP6, Slo1‐β4, and Slo1‐γ1. Interestingly, their binding sites are all different. We also present some examples of how functionally critical binding sites can be determined by introducing mutations. These structure‐guided approaches would be effective in understanding how VSD‐bound auxiliary subunits modulate ion channels.

Although the difference between the characteristics of fast and slow muscles has been extensively studied, it is still not fully understood. Here, we propose that nicotinic acetylcholine receptors (AChRs) expressed in slow muscles of zebrafish have high Ca2+ permeability compared to that of AChRs of fast muscles. To analyse the significance of the Ca2+ influx through AChRs in slow muscles, we generated a transgenic (Tg) zebrafish line that expresses Ca2+-impermeable AChRs in its slow muscles. The locomotor activities of the Tg zebrafish were markedly decreased at 1–3 days post-fertilization (dpf) compared to those of zebrafish expressing Ca2+-permeable AChRs in their slow muscles. Ca2+ imaging suggested that Ca2+ influx via AChRs is crucial for the Ca2+ response during muscle contraction in 2 dpf larvae, as slow muscle cells of the Tg line lacked a sustained Ca2+ response. Furthermore, we found that slow muscles of the Tg line became thinner compared to those expressing Ca2+-permeable AChRs. These short Ca2+ responses and thinner slow muscles may have induced locomotion impairment in the Tg line. These results suggested the physiological roles of the Ca2+ influx through AChRs in slow muscles and provided new insights into the characterization of fast and slow muscles.

In voltage-gated K + channels, membrane depolarization induces an upward movement of the voltage-sensing domains (VSD) that triggers pore opening. KCNQ1 is a voltage-gated K + channel and its gating behaviour is substantially modulated by auxiliary subunit KCNE proteins. KCNE1, for example, markedly shifts the voltage dependence of KCNQ1 towards the positive direction and slows down the activation kinetics. Here we identify two phenylalanine residues on KCNQ1, Phe232 on S4 (VSD) and Phe279 on S5 (pore domain) to be responsible for the gating modulation by KCNE1. Phe232 collides with Phe279 during the course of the VSD movement and hinders KCNQ1 channel from opening in the presence of KCNE1. This steric hindrance caused by the bulky amino-acid residues destabilizes the open state and thus shifts the voltage dependence of KCNQ1/KCNE1 channel. KCNQ1 is a voltage-gated K + channel and gating is modulated by auxiliary subunit KCNE proteins. Here, Nakajo and Kubo identify KCNQ1 phenylalanine residues in the voltage sensor and pore domains that are responsible for the gating modulation by KCNE1.

The KCNQ1 voltage-gated potassium channel and its auxiliary subunit KCNE1 play a crucial role in the regulation of the heartbeat. The stoichiometry of KCNQ1 and KCNE1 complex has been debated, with some results suggesting that the four KCNQ1 subunits that form the channel associate with two KCNE1 subunits (a 4∶2 stoichiometry), while others have suggested that the stoichiometry may not be fixed. We applied a single molecule fluorescence bleaching method to count subunits in many individual complexes and found that the stoichiometry of the KCNQ1 − KCNE1 complex is flexible, with up to four KCNE1 subunits associating with the four KCNQ1 subunits of the channel (a 4∶4 stoichiometry). The proportion of the various stoichiometries was found to depend on the relative expression densities of KCNQ1 and KCNE1. Strikingly, both the voltage-dependence and kinetics of gating were found to depend on the relative densities of KCNQ1 and KCNE1, suggesting the heart rhythm may be regulated by the relative expression of the auxiliary subunit and the resulting stoichiometry of the channel complex.

The KCNE family (KCNE1-5) is a group of single transmembrane auxiliary subunits for the voltage-gated K + channel KCNQ1. The KCNQ1-KCNE complexes are crucial for numerous physiological processes including ventricular repolarization and K + recycling in epithelial cells. We identified a new member of the KCNE family, “KCNE6”, from zebrafish. We found that KCNE6 is expressed in the zebrafish heart and is involved in cardiac excitability. When co-expressed with KCNQ1, KCNE6 produces a slowly activating current like the slow delayed-rectifier K + current (I Ks ) induced by KCNE1, despite the fact that the KCNE6 amino acid sequence has the highest similarity to that of KCNE3, which forms a constitutively open channel with KCNQ1. The kcne6 nucleotide sequences exist throughout vertebrates, including humans, although only the KCNE6 proteins of lower vertebrates, up to marsupials, can modulate KCNQ1, and it has become a pseudogene in eutherians. Our findings will facilitate a better understanding of how the KCNE family has evolved to modulate KCNQ1. Kasuya et al. identified a novel KCNE family member, KCNE6, in zebrafish, which is expressed in the heart and contributes to cardiac excitability by producing a slowly activating current with KCNQ1, similar to the I Ks current. This discovery enhances understanding of the evolutionary role of KCNE family members in modulating KCNQ1 and provides insights into the physiological diversity of potassium channel regulation across vertebrates.

In many mammalian neurons, dense clusters of ion channels at the axonal initial segment and nodes of Ranvier underlie action potential generation and rapid conduction. Axonal clustering of mammalian voltage-gated sodium and KCNQ (Kv7) potassium channels is based on linkage to the actin-spectrin cytoskeleton, which is mediated by the adaptor protein ankyrin-G. We identified key steps in the evolution of this axonal channel clustering. The anchor motif for sodium channel clustering evolved early in the chordate lineage before the divergence of the wormlike cephalochordate, amphioxus. Axons of the lamprey, a very primitive vertebrate, exhibited some invertebrate features (lack of myelin, use of giant diameter to hasten conduction), but possessed narrow initial segments bearing sodium channel clusters like in more recently evolved vertebrates. The KCNQ potassium channel anchor motif evolved after the divergence of lampreys from other vertebrates, in a common ancestor of shark and humans. Thus, clustering of voltage-gated sodium channels was a pivotal early innovation of the chordates. Sodium channel clusters at the axon initial segment serving the generation of action potentials evolved long before the node of Ranvier. KCNQ channels acquired anchors allowing their integration into pre-existing sodium channel complexes at about the same time that ancient vertebrates acquired myelin, saltatory conduction, and hinged jaws. The early chordate refinements in action potential mechanisms we have elucidated appear essential to the complex neural signaling, active behavior, and evolutionary success of vertebrates.

Although the difference between the characteristics of fast and slow muscles has been extensively studied, it is still not fully understood. Here, we propose that nicotinic acetylcholine receptors (AChRs) expressed in slow muscles of zebrafish have high Ca 2+ permeability compared to that of AChRs of fast muscles. To analyse the significance of the Ca 2+ influx through AChRs in slow muscles, we generated a transgenic (Tg) zebrafish line that expresses Ca 2+ -impermeable AChRs in its slow muscles. The locomotor activities of the Tg zebrafish were markedly decreased at 1–3 days post-fertilization (dpf) compared to those of zebrafish expressing Ca 2+ -permeable AChRs in their slow muscles. Ca 2+ imaging suggested that Ca 2+ influx via AChRs is crucial for the Ca 2+ response during muscle contraction in 2 dpf larvae, as slow muscle cells of the Tg line lacked a sustained Ca 2+ response. Furthermore, we found that slow muscles of the Tg line became thinner compared to those expressing Ca 2+ -permeable AChRs. These short Ca 2+ responses and thinner slow muscles may have induced locomotion impairment in the Tg line. These results suggested the physiological roles of the Ca 2+ influx through AChRs in slow muscles and provided new insights into the characterization of fast and slow muscles.

Tetrameric voltage-gated K+ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K+ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel at physiological voltages by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the KCNQ1-KCNE3-calmodulin complex structure, we thoroughly surveyed amino acid residues on KCNE3 and the S1 segment of the KCNQ1 voltage sensor facing each other. By changing the side-chain bulkiness of these interacting amino acid residues, we found that the distance between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of KCNQ1 and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests that tight binding of the S1 segment and KCNE3 is crucial for controlling the voltage sensor domains.